Data Sheet 1_Cyclic di-AMP alleviates periodontitis by activating PI3K/Akt/Nrf2 pathways.docx

Emerging research demonstrates the regulatory effects of c-di-AMP, a bacterial-derived small molecule secondary messenger, on host immune responses and promoting resistance against infection-related diseases. This study aims to elucidate the role of c-di-AMP in the occurrence and development of periodontitis. Using model of ligation-induced periodontitis, we observed that c-di-AMP effectively alleviated alveolar bone resorption. Transcriptomic sequencing in mice gingival tissues demonstrated that treatment with c-di-AMP led to a significant upregulation of the PI3K/Akt signaling pathway and its key components, including Akt3. Concurrently, we observed an upregulation of the cGMP/PKG signaling pathway. To validate our findings, we treated gingival epithelial cells with c-di-AMP and confirmed the activation of the PI3K/Akt pathway by c-di-AMP in gingival epithelial cells. Under LPS-induced inflammation, c-di-AMP significantly suppressed the release of inflammatory factors (such as IL-6 and TNF-α) from gingival epithelial cells. Moreover, key components of the PI3K/Akt pathway, including Akt, and downstream inflammation regulatory gene Nrf2, were upregulated, which were also confirmed at the protein level. Collectively, this study demonstrates that c-di-AMP definitely plays a role in alleviating periodontitis. Our findings highlight the mechanisms by which c-di-AMP modulates periodontitis, including activating the PI3K/Akt pathway and potentially involving the cGMP/PKG pathway, ultimately contributing to improved immune defense and maintenance of bone homeostasis.新兴研究表明，细菌来源的小分子第二信使环二腺苷酸（c-di-AMP）可调控宿主免疫反应，并增强机体抵御感染性疾病的能力。本研究旨在阐明环二腺苷酸在牙周炎发生与发展过程中的作用。本研究采用结扎诱导性牙周炎模型，观察到环二腺苷酸可有效缓解牙槽骨吸收。对小鼠牙龈组织开展转录组测序后发现，经环二腺苷酸处理后，磷脂酰肌醇3-激酶/蛋白激酶B（PI3K/Akt）信号通路及其关键组分（包括Akt3）的表达显著上调。与此同时，本研究还观察到环磷酸鸟苷/蛋白激酶G（cGMP/PKG）信号通路的表达出现上调。为验证上述研究结果，本研究用环二腺苷酸处理牙龈上皮细胞，证实其可在牙龈上皮细胞中激活PI3K/Akt通路。在脂多糖（LPS）诱导的炎症环境中，环二腺苷酸可显著抑制牙龈上皮细胞释放炎性因子（如IL-6、TNF-α）。此外，PI3K/Akt通路的关键组分（包括Akt）以及下游炎症调控基因核因子E2相关因子2（Nrf2）的表达均出现上调，且该结果在蛋白水平得到了验证。综上，本研究证实环二腺苷酸可切实缓解牙周炎。本研究结果揭示了环二腺苷酸调控牙周炎的潜在机制：通过激活PI3K/Akt通路，并可能涉及cGMP/PKG通路，最终改善机体免疫防御能力并维持骨稳态。